The human cytochrome P450s collectively metabolize a multitude of drugs and non-drug xenobiotics including toxins, mutagens and carcinogens as well as endobiotics such as steroids, prostaglandins and fatty acids. Drug metabolism in human liver is primarily catalyzed by twelve major microsomal P450 enzymes having different substrate and product specificities and are heterogeneously distributed in tissues (Ioannides, 1996, CYTOCHROMES P450 METABOLIC AND TOXICOLOGICAL ASPECTS. CRC Press, New York) (Rendic and DeCarlo, 1997, Drug Metab Rev 29:413-580). P450-catalyzed metabolism of drugs and non-drug xenobiotics is a key element in drug disposition and may be responsible for certain adverse drug reactions, chemical toxicity and immunotoxicity (Ioannides, 1996, CYTOCHROMES P450 METABOLIC AND TOXICOLOGICAL ASPECTS. CRC Press, New York).
The human cytochrome P450 2C sub-family consists of four isoforms 2C8, 2C9, 2C18 and 2C19. The three major alleles of P450 2C9 are the wild type 2C9Arg144(*1), 2C9Cys144(*2), 2C9Ile→Leu359(*3) (Haining et al. 1996, Miners and Birkett, 1998, Clin Pharmacol 45:525-538; hereinafter referred to as 2C9*1, 2C9*2 and 2C9*3 respectively). The 2C isoforms are collectively among the most important human enzymes responsible for the metabolism of a wide variety of drugs including taxol (Rahman et al., 1994, Cancer Res 54:5543-5546) phenytoin, tolbutamide, S-warfarin, losartan, S-mephenytoin and diazepam (Miners and Burkett, 1998, Clin Pharmacol 45:525-538).
Monoclonal antibodies (“MAbs”) are reagents (Yelton and Scharff, 1981, Annu Rev Biochem 50:657-680) that have proved to be of great value for the precise identification, measurement and functional characterization of each P450 isoform (Gelboin, 1993, Pharmacol Rev 45:413-453). The MAbs are derived from potentially immortal hybridomas and are specific and highly inhibitory to the enzyme activity of the target P450 and thus are powerful reagents for “reaction phenotyping” i.e., for measuring the metabolic contribution of each of the multiple P450s to a substrates metabolism (Gelboin, 1993, Pharmacol Rev 45:413-453)
MAbs specific to seven individual major human liver (HLM) P450 isoforms were described viz., 1A1, (Fujino et al., 1982, Proc Natl Acad Sci USA 79:3682-3686), 1A2 (Yang et al., 1998, Pharmacogenetics 8:375-382), 2A6 (Sai et al., 1999, Pharmacogenetics 9:229-237), 2B6 (Yang et al., 1998, Biochem Pharmacol 55:1633-1640), 2D6 (Gelboin et al., 1997, Pharmacogenetics 7:469-477), 2E1 (Gelboin et al., 1996, Chem Res Toxicl 9:1023-1030) and 3A4/5 (Gelboin et al., 1995, Biochem Pharmacol 50:1841-1850) and to the entire 2C sub-family (Yang et al., 1998, Biochem Pharmacol 55:889-896; Park et al., 1989, Biochem Pharmacol 38(18):3067-3074). The MAbs measured the contribution of each target P450 to the metabolism of a variety of examined drugs (Yang et al., 1999, Drug Metab Dispos 27:102-109; Gelboin et al., 1999, Trends Pharmacol Sci 20(11):432-438). Many of the MAbs can also be used to measure the tissue P450 protein content by immunoblot analysis.
Genetic polymorphisms of cytochromes P450 result in phenotypically-distinct subpopulations that differ in their ability to perform biotransformations of particular drugs and other chemical compounds. These phenotypic distinctions have important implications for selection of drugs. For example, a drug that is safe when administered to most humans may cause toxic side-effects in an individual suffering from a defect in an enzyme required for detoxification of the drug. Alternatively, a drug that is effective in most humans may be ineffective in a particular subpopulation because of lack of a enzyme required for conversion of the drug to a metabolically active form. Further, individuals lacking a biotransformation enzyme are often susceptible to cancers from environmental chemicals due to inability to detoxify the chemicals (Eichelbaum et al., 1992, Toxicology Letters 64/65, 155-122). Accordingly, it is important to identify individuals who are deficient in a particular P450 enzyme, so that drugs known or suspected of being metabolized by the enzyme are not used, or used only with special precautions (e.g., reduced dosage, close monitoring) in such individuals. Identification of such individuals may indicate that such individuals be monitored for the onset of cancers.
Existing methods of identifying deficiencies in patients are not entirely satisfactory. Patient metabolic profiles are often assessed with a bioassay after a probe drug administration. Poor metabolizers (PM) exhibit physiologic accumulation of unmodified drug and have a high metabolic ratio of probe drug to metabolite. This bioassay has a number of limitations: Lack of patient cooperation, adverse reactions to probe drugs, and inaccuracy due to coadministration of other pharmacological agents or disease effects. See, e.g., Gonzalez et al., 1994, Clin. Pharmacokin. 26, 59-70. Genetic assays by RFLP (restriction fragment length polymorphism), ASO PCR (allele specific oligonucleotide hybridization to PCR products or PCR using mutant/wild-type specific oligo primers), SSCP (single stranded conformation polymorphism) and TGGE/DGGE (temperature or denaturing gradient gel electrophoresis), MDE (mutation detection electrophoresis) are time-consuming, technically demanding and limited in the number of gene mutation sites that can be tested at one time.
A complication in patient drug choice is that most drugs have not been characterized for their metabolism by P450 2C family and other cytochromes P450. Without knowing which cytochrome(s) P450 is/are responsible for metabolizing an individual drug, an assessment cannot be made for the adequacy of a patient's P450 profile. For such drugs, there is a risk of adverse effects if the drugs are administered to poor metabolizers.
Monoclonal antibodies that specifically bind to 2C family members and inhibit its activity, if available, could be used to screen drugs for their metabolism by 2C and/or identify 2C poor metabolizers by simple bioassays, thereby overcoming the problems in prior complicated methods discussed above. However, such monoclonal antibodies represent, at best, a small subset of the total repertoire of antibodies to human cytochrome P450 2C, and have not hitherto been isolated. Although in polyclonal sera, many classes of antibody may contribute to inhibition of enzyme activity of P450 2C family members as a result of multiple antibodies in sera binding to the same molecule of enzyme, only a small percentage of these, if any, can inhibit as a monoclonal. A monoclonal antibody can inhibit only by binding in such a manner that it alone block or otherwise perturb the active site of an enzyme. The existence and representation of monoclonal antibodies with inhibitory properties thus depend on many unpredictable factors. Among them are the size of the active site in an enzyme, whether the active site is immunogenic, and whether there are any sites distal to the active site that can exert inhibition due to stearic effects of antibody binding. The only means of obtaining antibodies with inhibitory properties is to screen large numbers of hybridoma until one either isolates the desired antibody or abandons the task through failure.
Notwithstanding these difficulties, the present invention provides inter alia monoclonal antibodies that specifically bind to human cytochrome P450 2C family members and inhibit their activity.